Field of Invention
The present invention relates generally to a simple and low cost diagnostic device for single cell separation and analysis. More specifically, the present invention relates to a microfiltration platform having a well-defined microsieve capable of separating and capturing target cells from a fluid sample for rapid interrogation in assessing cell status or diagnosing disease.
Description of Related Art
Single cell technologies are of extreme importance when only very few events are present in a sample. Examples of these are bacteria in bodily fluids and circulation tumor cells (CTC) in blood. By collecting these single events and subsequently perform analysis on the collected events such as analyzing DNA mutations and RNA/protein expression at a single cell level, a signature for these events can be established leading to a more specific treatment, development of new treatments and understanding of the underlying biological processes.
One common method to isolate cells for single cell analysis is by mechanically separating the cells into wells. Depending on the intended application a microwell device can be designed in numerous ways and with numerous different materials. Well-shaped structures of 10 and 20 μm in diameter have been fabricated using PDMS stamping of PEG poly(ethylene glycol) onto silicon substrates (Suh et al., 2004), and polystyrene substrates (Dusseiller et al., 2005). Mid-sized wells have been fabricated by surface engineered PEG on glass, creating arrays for improved optical cell imaging with wells capable of harboring more than one cell, such as 30×30 μm (Revzin, 2003) or 15×15 μm (Revzin et al., 2005) wells.
Suspensions of single cells are normally seeded manually into microwells, and the cells are randomly positioned in the wells by gravitation/sedimentation. To minimize the chance of having multiple cells within a single well, cell suspensions are diluted, causing a low percentage of wells actually filled. Other methods for seeding single cells into individual wells require wells with a volume that can only hold a single cell, eliminating the ability to add additional reagent to individual wells.
Thus the application of these designs in diagnostics is severally limited due, in part, because the remaining cells outside the well are flushed away, sometimes followed by another round of cell loading to increase the final number of captured cells. In cases where only a limited number of events (or cells) are present, as for example in the analysis of CTC, it would be detrimental to have cells outside the wells where they are flushed away.
Larger wells require micromanipulation to retrieve the cells from the wells. An example of cell retrieval from smaller cell-sized wells using micromanipulation was demonstrated by Tokimitsu et al., 2007. In general, cell retrieval and/or removal are important aspect for microwell chip design. However many single-cell micro-chips are designed to provide analysis with a continuous flow across the chip without the possibility for the investigator retrieving cells or clones to further analyze. Techniques for retrieval and manipulation of cells are very important, since sample screening often involves only a few cells worthy of further detailed analysis.
Filtration membranes in a microfiltration platform provide a means for capturing target events within a sample. Critical factors that determine a microfiltration platform in a diagnostic device are membrane composition and fluidic pathway design for liquid and sample manipulation. It is known that membrane filters are an indispensable necessity in the field of diagnostics such as in sample preparations for scanning electron micrographs where track etched membranes are used or in determining the number and type of micro-organisms and/or cells in a given sample.
Micromachined microsieves have been described as a type of microfiltration membrane comprising a supporting substrate and a thin membrane layer with precisely etched pores which are mechanically stable and have high pressure strength at a thickness of only a few hundred nanometers. Thus, these microsieves are useful for diagnostic applications and have been incorporated, in part, in the present invention. Prior to the present invention only conventional filtration membranes were used. With respect to current filtration membranes microsieves have several specific advantages including, in part, a very low flow resistance, regular and precise pore geometry and an optically flat surface. The sample liquid is filtered through the microsieve which has a low flow resistance allowing for high flow rates which results in the collection of cells and microorganisms in a relatively short time. The optically flat surface enables a single image of the microsieve surface to be acquired without the need to refocus on different locations across the microsieve. Furthermore the microsieve is chemically inert and has no disadvantageous fluorescence back light scattering which further improves the staining and detection of micro-organisms for imaging through a fluorescence microscope.
Polymeric materials currently used in conventional filtration membranes are not well suited as microsieves. Membranes formed with these materials are characterized by relatively small values for Young's Modulus and/or a low yield strength and so are not suitable for fabricating into microsieves.
Another problem associated with the use of current filtration membranes to capture cells or particles from fluids is the inability of the sample fluid to easily start flowing through the openings in a microsieve membrane. Most micromachined filters have an inorganic membrane layer such as silicon nitride or silicon oxide with water contact angles above 30° and in time can even rise above 60°. At a pore size of 1 um, fluid flow through the microsieve can then only be induced at pressures above 100 mbar. A normal procedure to reduce this pressure is to create hydrophilic hydroxyl groups with oxygen plasma at the membrane surface just before use. Another normal procedure to reduce this pressure is to pre-wet the back side of the microsieve with an additional fluid. However for many applications, especially for in vitro diagnostic point of care analysis, the pressure needs to be reduced to zero. At zero pressure the fluid will flow through the filter without the need of applying pressure. Without the need to pre-wet or pressurize the fluid, the microsieves become usable in a wide range of applications where they were rather unpractical before.
Accordingly with these limitations in the prior art, there exists a medical need to develop a filtration platform which incorporates the micromachined microsieves described herein,